Coating and coating method

ABSTRACT

The present invention discloses a coating for a medical implant, wherein at least a part of said coating contains an osseointegration agent and the same and/or a different part of the coating contains an antimicrobial metal agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Stage patent application filed under 35 U.S.C.§371 from PCT International Application Number PCT/US2009/035467,International Filing Date 27 Feb. 2009, International Publication NumberWO 2009/111307, International Publication Date 11 Sep. 2009, which isincorporated herein by reference in its entirety, which PCT application,and this application, claim priority to U.S. Provisional PatentApplication Ser. No. 61/032,621, filed 29 Feb. 2008, and to U.S.Provisional Patent Application Ser. No. 61/051,783, filed a9 May 2008,both of which U.S. Provisional Patent Applications are incorporatedherein by reference in their entireties.

The present invention relates generally to coating processes and moreparticularly to coating processes for implantable prosthesis formineralized body parts.

BACKGROUND OF THE INVENTION

Medical implants for use in human and animal bodies is known in the artsto serve numerous purposes, both short-term and long-term. One commoncomplication associated with medical implants is infection at theimplantation site. Implant related infection is managed by a number ofdifferent means, including use of prophylactic systemic antibioticsadministered to the patient during set periods before and afterimplantation surgery. However, infection of the host site remains acommon problem, most often requiring a second surgery to remove theimplant. The reason for implant removal is that when an infectionoccurs, the implant acts as the site preferred by the invading bacteriato colonize. When bacterial floras thus colonize the implant, they areextremely resistant to eradication via delivery of antibiotic drugsthrough systemic means (such as oral or intravenous). The only means tomanage such an infection is revision surgery to remove the colonizedimplant, to be replaced by another implant system. Revision surgery isassociated with all of the complications of any surgery, which includeinfection. Additionally, the patient body now has to deal withmorbidities of two surgeries. Other undesirable aspects of revisionsurgery include blood loss and thrombosis. Revision surgery is alsoassociated with greater risk of implant failure, and the medicalprognosis of a revision surgery is never as good as primary surgery.Revision surgery is more costly than primary surgery, and it requiresadditional longer recovery time for the patient to become fullyfunctional, which can result in loss of productivity. As can be seen inthe in the preceding, revision surgery due to implant site infection isa major medical problem for the patient, and a major drain on the costof treatment. A technology that reduces or eliminates implant relatedinfection will make an important positive contribution to the patient'swell being and the economics of delivering medical care.

It is well known in medicinal arts that the metal silver and itscompounds are good antibacterial agents. As such, silver and itscompounds are routinely used to treat superficial skin infections.Silver compounds are typically administered to a new born baby's eyes toward off potential infections. One major advantage of silver compoundsover traditional antibiotic drugs is that infectious bacteria do notform resistance to silver. Drug resistant bacteria are a major cause forconcern in the field of treating infections. Silver compounds,therefore, have the potential for preventing bacterial infections inmedical implants, without the risk of developing resistant strains ofbacteria.

Numerous attempts have been made in the past to incorporate metallicsilver and/or its compounds into the outer surfaces of medical implants.These attempts and limitations thereof are summarized below.

Silver can be plated (coated) onto medical implants by any number ofmeans known in the arts, including electroplating, electro-deposition,and painting with an organic polymer carrier. Other techniques forcoating include chemical vapor deposition (CVD) and physical vapordeposition (PVD). CVD and PVD techniques are very expensive and requirehighly sophisticated, controlled manufacturing processes. All of thesecoating techniques have limited use in implants because the biologicalinterface, i.e., the interface between the implant and the host tissueis completely different for the coated implant versus the uncoatedimplant. This limitation can have a profound impact on the success ofthe medical implant, whose biological interfact is usually engineeredfor integration with the host tissue. This need for the implantbiological interface to have antimicrobial properties simultaneous withtissue integration properties forms an important aspect of thisinvention as will be shown later.

Attempts have been made to “implant” ions of silver onto surfaces ofmedical implants. This technique utilizes energetic beams (highvelocity) of ionized silver which are impinged upon the surface of theimplant. This technique is referred to as “ion implantation”. Silver ionimplantation affects only a very thin surface layer (typically in therange of nanometers thick), and therefore has limited use for long-termeffectiveness against infectious agents. The “implanted” silver ions maybe so well incorporated into the medical implant surface that verylittle would leach out into the surrounding tissue for effective kill ofbacteria. Similar limitations are present in the technique called “ionbeam assisted deposition” (IBAD). Ion implantation techniques are alsovery expensive and require sophisticated, controlled manufacturingprocesses.

It was discussed previously that the biological interface of theimplant, effectively the surface of the implant in contact with hosttissue, is typically engineered to integrate with the host tissue. Acommon surface engineering technology used in medical implants,particularly bone contacting orthopaedic, spinal, and dental implants ishydroxyapatite (HA) coating.

Hydroxyapatite (HA) coatings are applied to medical implants using anumber of different methods, including plasma spray, electrodeposition,solution precipitation, and sol-gels methods. Attempts have also beenmade to incorporate silver into HA. Mainly, the prior art has used thefollowing two different methods to provide a silver-doped HA coating:

One method of incorporating a silver derivative into HA includes thesteps of sequentially applying layers of stable silver oxide and HApowder to an implant. However, this method does not form a homogeneoussilver-doped HA coating. Consequently, coverage is not uniform, and ionrelease is not steadily maintained after implantation. Even if thesilver oxide is mixed with the HA powder prior to plasma-spraying, theoxide cannot chemically react and combine with conventional HA powder toform a homogenous formulation.

The second method has been to soak an implant having a hydroxyapatitecoating in a silver nitrate solution for approximately 24 hours and thendry the implant, in air or an inert environment. This method relies onapplying silver into an implant that has already been coated with HA.This post hoc method has the potential of perturbing the physical andmechanical characteristics of the existing HA coating, which may not bedesirable from the stand-point of HA attachment to the substrate medicalimplant. Additionally, this technique does not allow sufficient silvernitrate to soak deep into the HA coating. HA coatings on medicalimplants can range in thickness from a few to many hundreds of microns.Incorporation of silver by this method requires an ion exchange reactionbetween silver nitrate and HA coating. This ion-exchange reaction onlyoccurs at the outer surface, where the HA coated implant makes contactwith silver nitrate solution. Therefore, although silver is incorporatedthroughout the HA matrix at the surface, it is not incorporated into theHA matrix at the full thickness of the HA coating. Because silver isincorporated only at the surface and near surface region of the HAcoating (and not throughout the thickness), the release of silver ionsinto the host tissue is likely to be shortlived; that is release ofsilver ions will not be sustained for very long periods required toward-off infections a few months after implantation Moreover, the ionexchange reaction requires careful control of the pH of the silvernitrate solution. The ideal pH of the silver nitrate solution may not beamenable to preservation of the HA structure already coated on theimplant. In such a method, the HA coating attachment to the substratemedical implant may be compromised during the soaking process if the pHof the silver salt solution is not controlled.

The preceding discussion shows the limitations of prior art methods ofincorporating silver and its compounds into the biological interfaces ofmedical implants. There is a need for commercially viable means ofachieving antimicrobial coatings of medical implants. The ideal medicalimplant with antimicrobial properties would be such that themanufacturing of the same would not deviate from currently practicedmanufacturing processes such as plasma spray.

SUMMARY OF THE INVENTION

In one embodiment of the invention there is provided a coating suitablefor a medical implant. Such implants may have beneficial effects, suchas they may be able to promote osseointegration and/or simultaneouslyreduce or eliminate the risk of infection at the implant site.

According to some embodiments, there is provided a coating for a medicalimplant, in which at least a part of said coating contains anosseointegration agent and the same and/or a different part of thecoating contains an antimicrobial metal agent.

According to some embodiments of the present invention, the medicalimplant has a coating that interfaces with the host biologicalenvironment. Such a coating preferably comprises an agent which promotesosseointegration or osteoconductivity, such as a calcium derivative. Theterm “osseointegration agent” as used herein refers to any agent thathas the capability of encouraging the integration of an implant withbone. The term “osteoconductivity” refers to the situation where theimplant is able to support the attachment of new osteoblasts andosteoprogenitor cells, providing a structure through which new cells canmigrate and new vessels can form.

It is to be understood that the term “calcium derivative” as usedthroughout the description is used as a representative term for an agentthat may promote osseointegration or osseoconductivity, and isfrequently referred to as hydroxyapatite (HA), but can be any suitablederivative, examples of which the skilled person is well aware. Oneexample is HA, but other derivatives such as calcium phosphate, calciumorthophosphate, tricalcium phosphate, ceramic bioglass can all serve thefunction of this invention, and are incorporated herein withoutlimitation. Other forms of surface engineering to enhance integration ofthe implant to host tissue include coatings apatites such as calciumphosphate, hydroxyapatite, β tricalcium phosphate, a mixture ofhydroxyapatite and β tricalcium phosphate, resorbable polymers,bioglass, derivatised phosphate-based compound, orthophosphates,monocalcium phosphates, octacalcium phosphates, dicalcium phosphatehydrate (brushite), dicalcium phosphate anhydrous (monetite), anhydroustricalcium phosphates, whitlocktite, tetracalcium phosphate, amorphouscalcium phosphates, fluoroapatite, chloroapatite, non-stoichiometricapatites, carbonate apatites, biologically-derived apatite, calciumhydrogen phosphate, calcium hydrogen apatite, water insoluble ceramics,phosphates, polyphosphates, carbonates, silicates, aluminates, borates,zeolites, bentonite, kaolin, and combinations thereof. These techniquesof surface engineering are incorporated herein without limitation.

In some embodiments, the calcium derivative is one, or a combination, ofhydroxyapatite and/or β tricalcium phosphate.

According to some embodiments of the present invention, theantibacterial efficacy of the native calcium derivative (e.g. HA)coating is improved by the addition of silver.

The silver may be present as one or more of a silver-substituted calciumderivative (such as HA) and/or discrete metallic silver particles.

Preferably, the antimicrobial metal agent is present in at least a partof said coating as discrete particles. The antimicrobial metal maycomprise one or more of silver, copper, and/or zinc. The discreteparticles (e.g. silver particles) may preferably be distributedthroughout the entire coating thickness.

The present inventors have found that it is preferable to have metallicsilver incorporated into the medical implant rather than a discretecompound of silver (such as silver oxide).

The use of silver (or other antimicrobial metallic species such ascopper) can be engineered in a number of ways. Because the metallic antimicrobial is to be used in host animal (including human) tissue, itsproperties should serve the following non-limiting important functions:

(a) The antimicrobial treated medical implant should resist bacterialcolonization. The chemical form of silver present in the coating caninfluence this. For instance, silver can be present in the coating onthe implant surface as a chemical compound, such as silver phosphate,silver oxide, silver nitrate, and other compounds. Silver can also bepresent in the metallic silver form. Additionally, a mixture of silvercompounds and/or metallic silver can be present. The exact compositionwill influence the ability of the implant to resist microbialcolonization upon implantation, and over time as the antimicrobialcoating reacts in the host tissue environment.

(b) The antimicrobial treated medical implant should release theantimicrobial metal into the host tissue to affect its antimicrobialproperties. The antimicrobial metal can be released into the host tissueas ions of silver or as metallic silver, or as a compound of silver(e.g., silver phosphate or silver oxide). The chemical nature ofantimicrobial release will depend on the composition of the coating. Itis appreciated that the preferred type of release (ionic, metallic, orcompound) can be controlled by the composition of the antimicrobialcoating.

(c) The release kinetics of silver into the host environment may be inthe form of a burst release (bolus) or sustained over a long period oftime or a combination of burst and sustained release. The releasekinetics and duration of release can be engineered by the selection ofthe chemical nature of the silver and its compounds in the coating, asdiscussed above.

(d) The release of silver in the forms discussed above should be indoses that are well tolerated by the host living tissue, organ, andorganism. The release should not interfere to appreciable degree withother biological and mechanical functions of the implant and thecoating. The coating and the release of silver should be biocompatibleand should not present a cytotoxic challenge to the host environment.This can be controlled, as listed above.

The type and concentration of silver and its compounds present in theantimicrobial coating can be controlled by any number of means. Silvercan be incorporated into coating particulate (such as HA) by an ionexchange reaction with silver nitrate or other silver compound as knownin the arts. After the ion exchange reaction, the excess silver compoundcan be completely rinsed out of the HA powder, or just partially rinsed.Therefore the HA powders can contain no unreacted silver compounds orcan retain all of the unreacted excess silver compounds, or can retainpartial quantities of unreacted silver.

In some embodiments, the discrete metal particles (e.g. metallic silverparticles) are present in a coating having an osseointegration agentthat is not itself substituted with an antimicrobial metal agent.Optionally, the metal particles are distributed in a coating having anosseointegration agent which has also been substituted with anantimicrobial metal agent. Preferably the metal agent is silver and ispresent in the coating as silver substituted into the osseointegrationagent.

Optionally, one method of providing a coating to a medical implant is bya technique called “plasma spray”. The thickness of the plasma sprayedcoating on a medical implant can typically be from about 1 micron, andtypically is in the range of 10 or a few tens to a few hundreds ofmicrons. Coating thickness in excess of a few thousand microns (onemillimeter or greater) are also possible. Other methods of applyingcalcium-derivative coatings onto medical implants include sol-gelmethods, electrodeposition, solution precipitation, and biomimeticcoatings.

According to some embodiments of the present invention the implant isplasma-sprayed with a powder of an osseointegration agent (e.g. calciumderivative such as HA). Preferably, the osseointegration agent is atleast partially substituted with an antimicrobial metal agent (e.g.silver) prior to plasma spraying.

Plasma spray coating can be applied under atmospheric conditions orunder reducing conditions, or in a vacuum. Additionally, the plasmaspray equipment can be under controlled environmental conditions. Forinstance the environment may contain argon, or other inert gases. Or theenvironment may contain reactive gases. The environment under which theplasma spray equipment is operated under can determine the type ofsilver (or compound thereof) that is incorporated into the HA coating.For instance, atmospheric (uncontrolled environment) HA coating can leadto oxidation of silver (and compounds thereof) already present in the HAparticles via the ion exchange reaction as they exit the plasma andbefore they are coated onto the implant surface. Vacuum plasma sprayingcan ameliorate this oxidation reaction. If specific silver compounds aredesirable, then the environment of the plasma spray equipment can becontrolled to affect production of such compounds. If, for instance itis desirable to have silver fluoride as the silver compound in the HAcoating, then the environment can be enriched in fluorine by any numberof means known in the arts.

In one embodiment, no post-process heat treatment is required to furtherconsolidate the coating or bond it to the substrate.

If plasma spraying is to be utilised in the present invention, thepreferably the plasma spraying of at least one layer of the coatingwhich contains an antimicrobial agent (such as silver) is conducted in areducing environment.

Thus, in one embodiment of the invention there is provided a plasmasprayed coating for a medical implant, in which at least a part of saidcoating contains an osseointegration agent and the same and/or adifferent part of the coating contains an antimicrobial agent.

Preferably the osseointegration agent is a calcium derivative.

Preferably at least a part of the coating containing the antimicrobialagent has been plasma sprayed under reducing conditions, and preferablyplasma spraying is conducted in a vacuum.

Without wishing to be bound by theory, it is believed that when powderssuch as HA contain unreacted silver compounds, these compounds willdissociate into metallic (elemental) silver in the highly reducingenvironment (hydrogen and inert gas mixture) of the plasma. Therefore,if the plasma spraying is conducted in a vacuum condition, the reducedmetallic silver will be coated onto the medical implant as discretemetallic particles along with the HA and silver-containing HA. On theother hand if the plasma spray process was conducted under atmosphericconditions, the metallic silver formed in the reducing atmosphere of theplasma will react with oxygen to form an oxide. These oxides then arecoated onto the medical implant along with HA and silver containing HA.It can be appreciated from the previous discussion that the type ofsilver present on the medical implant can be controlled by the processof applying the coating onto the medical implant.

In some embodiments of the present invention, at least a part of saidcoating contains discrete particles of an antimicrobial metal. In someembodiments, said antimicrobial metal comprises one or more of metallicsilver, copper, and/or zinc.

Preferably the antimicrobial metal comprises metallic silver. In someembodiments, the metallic silver particles are spherical or irregular inshape. Further, the diameter of the metallic silver particles range fromabout 15 nm to about 10 μm in size.

In some embodiments of the invention, the silver concentration issufficient to have an anti-bacterial effect, such as having aconcentration from about 0.1 to about 10 weight percent.

In some embodiments, said calcium derivative contained in said at leasta part of said coating containing a calcium derivative issilver-substituted, and preferably that silver-substituted calciumderivative has a silver concentration that is homogenously distributedthroughout the thickness of the coating. The silver-substituted calciumderivative may contain from about 0.1 to about 10% by weight of silver,preferably from about 0.5 to about 3.0% by weight of silver.

In some embodiments, the at least a part of said coating that containsthe antimicrobial agent has the agent distributed throughout the entirethickness of the part of the coating.

With careful control of the manufacturing process, then, it is possibleto create plasma sprayed HA coated medical implants, which contain anyone or a combination of two or more of the following types of silver inthe HA coating;

(1) The silver can be present up to a fixed depth from the outer surfaceof the HA coating, or it can be present at the full thickness of the HAcoating.

(2) The silver can be in the form of a silver compound (e.g., silverphosphate, silver oxide) that is fully reacted with the HA andhomogeneously distributed throughout the HA coating. Alternatively thesilver compound can be distributed discretely into the matrix of the HAcoating.

(3) The silver can be fully incorporated into the native HA crystalstructure as silver-modified HA, and homogeneously distributedthroughout the HA coating,

(4) The silver can be in the form of metallic silver that fomis in thereducing atmosphere of the plasma. Such silver is distributed asdiscrete particles of silver in the matrix of the HA coating. Themetallic silver can be present up to a fixed depth from the outersurface of the HA coating, or it can be present at the full thickness ofthe HA coating.

In some embodiments of the invention, the coating is preferably greaterthan about 1 μm in thickness, preferably from about 10 μm to about 200μm, and most preferably from about 30 μm to about 100 μm. Said coatingis preferably well-adhered to the substrate material (e.g. Ti6A14V,cp-Ti, CoCrMo, Ta, and other biomedical materials) and possesses atensile bonding strength for example of at least 15 MPa.

According to some embodiments of the present invention, theosseointegration of native calcium derivative (e.g. HA) coating is notcompromised by the addition of silver.

In some embodiments of the present invention, the coating has one ormore osteoconductive, osteopromotive and/or antimicrobial properties.

According to some embodiments of the present invention the coating isformed from a homogeneous formulation of a calcium derivative (e.g. HA)powder containing silver. In some embodiments, no blending of thesilver-containing calcium-derivative powder with other silver-containingpowders is required to incorporate silver into the coating. For example,in one embodiment, no blending of HA powder with other powders (e.g.silver oxide powder) is required to incorporate silver into the HAcoating.

According to some embodiments of the present invention theosteointegration agent (e.g. HA) powder contains at least partiallyexcess silver reactant adsorbed onto the surface.

According to some embodiments of the present invention the HA powdercontains both silver substitution and excess silver reactants.

According to some embodiments of the present invention the silverreactant for substitution into osseointegration agent (e.g. HA) powderis one or more of a silver salt, such as silver nitrate (AgNO₃) and/orsilver fluoride (AgF). Alternatively, or in addition, the silverreactant can be one or more of a silver halide, such as silver iodide.

In some embodiments of the present invention, the calcium derivative, inaddition to silver, is also substituted with one or more of thefollowing: carbonate, fluoride, silicon, magnesium, strontium, vanadium,lithium, copper, and/or zinc.

According to some embodiments of the present invention, the silvercontaining calcium derivation (e.g. HA) powder is formed by firstsoaking conventional HA powder in a silver nitrate-containing and/orsilver fluoride-containing aqueous or organic solution for a period oftime. In some embodiments the aqueous or organic solution may compriseboth silver fluoride and silver nitrate.

It is preferred that the calcium derivative (e.g. HA) powder soaks andstirs in the solution for a suitable time to allow sufficient ionexchange reaction between HA powders and Ag Salt(s) solution. Such atime period can range from a few hours (e.g. 10 hours) to a week, suchas a period of approximately one day to three days. The reaction is morepreferably about two days. For best results, it may be necessary toavoid excessive exposure of the mixture to light.

The osseointegration agent (e.g. HA) powder may soak at roomtemperature, however, a slightly warmer temperature is preferred inorder to increase solubility of the solution into the HA. Thetemperature should not be elevated so much as to break down thecomposition.

It is also generally important to maintain a proper pH level of thesolution during the time in which the HA powder is reacting with AgSalts in the aqueous or organic solution. The pH should generally bekept at level such that there is a minimum of HA dissolution but at thesame time reduce the possibility that the silver nitrate will react withOH and precipitate to form silver hydroxide and eventually becomessilver oxide. The pH level may be maintained in the range of 6.5-8.5,but is preferably above 6.7 to prevent the HA from dissolving. Morepreferably, the pH level of the mixture is in the range of 6.8-7.2, butother levels may be acceptable.

After the ion exchange reaction, the mixture may be left to air dry ormay be rinsed and/or washed with deionized and distilled water (DDH₂O)and then allowed to air dry.

In some embodiments of the present inventions, there is provided amethod for preparing an antimicrobial coating, in which asilver-containing calcium derivative powder is produced by ion-exchangeor sol-gel methods and subsequently plasma sprayed onto a substratematerial.

In some embodiments, the silver-containing calcium derivative powdersare produced by: a. suspending calcium derivative powder in a silversalt solution for a time and temperature sufficient to exchange calciumions for silver ions, and b. drying said ion-exchanged and washedcalcium derivative.

In some embodiments, an additional step is performed between steps (a)and (b) of washing said ion-exchanged calcium derivative powders.

In some embodiments, the ion exchange reaction between the calciumderivative powder and silver salt solution occurs for about 24 to 168hours at a temperature of from about 20° C. to 95° C. In someembodiments, the silver salt solution is silver nitrate or silverfluoride with a concentration of 10⁻²-10⁻⁴ M and the mass ratio ofsilver nitrate to HA powder is within the range of 0.01-0.1.

Further, in some embodiments, the silver-containing calcium derivativepowders are produced by a sol-gel method by:

(a) Mixing a combination of calcium, silver, and/or phosphorusprecursors to obtain a homogenous sol-gel solution;

(b) Aging the sol-gel solution at the temperature between 20-95° C. fora suitable time period;

(c) Drying and calcining the sol-gel solution at a temperature aboveroom temperature for a suitable time period;

(d) Processing the calcined powders to a desired particle sizedistribution for subsequent plasma spraying process.

In some embodiments, the calcium precursor is calcium nitrate. In someembodiments, the silver precursor is silver nitrate. In someembodiments, the phosphorus precursor is ammonium dihydrogen phosphate.In some embodiments, the silver precursor concentration range is fromabout 0.1 wt % to 10 wt %, preferably from 0.5 wt % to 3.0 wt %.

In certain embodiments, fluorine and carbonate precursors are mixed withthe calcium, silver, and phosphorous precursors to obtain a homogenoussol-gel solution. In some embodiments, the fluorine precursor isammonium fluoride. In some embodiments, the carbonate precursor isammonium carbonate. In some embodiments, the F and/or carbonateprecursor concentration is about 10^(″2)-10^(″3)M.

In certain embodiments, carbonate, fluoride, silicon, magnesium,strontium, vanadium, lithium, copper, or zinc precursors, orcombinations thereof, are mixed with the calcium, silver, andphosphorous precursors to obtain a homogenous sol-gel solution.

The remaining solid mixture comprises a homogenous HA powder formulationhaving silver and/or fluoride therein. The homogenous HA powderformulation may or may not be ground up and then used in a conventionalplasma spray process to coat a biomedical implant or medical device aswould normal HA powder. The soaking and grinding process of the presentinvention generally only negligibly increases the mean particle size ofthe HA powder, and thereby does not interfere with subsequent plasmaspray processes that may be dependent on conventional equipment.

In some embodiments, the size distribution of the silver-containingcalcium derivative powder formulation prior to spraying is substantiallyequivalent to native pure calcium derivative powder.

One possible advantage of using the aforementioned air drying processwithout rinsing is that by not rinsing there may be a small amount ofexcess silver nitrate or silver fluoride in addition to the homogeneousHA/silver powder formulation after the aqueous/organic solutionevaporates. This might ensure that after the extremely high temperaturespresent during the plasma spraying process, some residual (i.e. “extra”)silver is present in the coating, even if some of the silver in thehomogeneous HA powder vaporizes. The silver content and degradationprofile in the final plasma sprayed coating can be tailored usingdifferent concentration of silver salts.

In one embodiment of the invention, there is provided a method oftreating a patient requiring a surgical implant, the method havingoperating on the patient, inserting a medical implant having a coatingof any of the embodiments described herein into the operating site, andleaving the implant in situ after the operation, in which the implantreduces the risk of, or eliminates, post-operative infection at the siteof the implant.

As used herein, the term “silver” may comprise substantially puresilver, a composition having a silver-based component, a precursorhaving a silver-based component, a silver compound, or an alloy havingsilver. From the preceding discussion, it is possible that silver and/orsilver compounds can be incorporated into the biological interface (suchas HA coating) of the medical implant, in such a manner that the surfacesimultaneously serves an antimicrobial function.

Other metallic species are also known to have similar antimicrobialeffects. These include, but are not limited to, copper, zinc, mercury,lead, and other metals. These metallic species have a wide range ofeffectiveness depending on the dose delivered. Additionally, some ofthese metallic species may be toxic to the host tissue, also dependingon the dose used and the tissue site in which they are applied. Althoughthis invention relates to silver as the preferred antimicrobial metallicspecies, it is not limited solely to silver. Mixtures of differentmetallic species can also be used. For instance varying amounts ofsilver and copper compounds can be used.

Further areas of applicability of the invention will become apparentfrom the detailed description provided hereinafter. It should beunderstood that the detailed description and specific examples, whileindicating the particular embodiment of the invention, are intended forpurposes of illustration only and are not intended to limit the scope ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the written description serve to explain theprinciples, characteristics, and features of the invention. In thedrawings:

FIG. 1 is a scanning electron micrograph of HA (a), Ag-HA-L (b), Ag-HA-M(c) and Ag-HA-H (d)-coated Ti6A14V discs.

FIG. 2 is a scanning electron micrograph of cross-sectional view of HA(left) and Ag-HA-H (right)-coated Ti6A14V discs. The silverconcentration was measured at different locations throughout the coatingfor the Ag-HA-H sample, as indicated by the “x” markers.

FIG. 3 shows tensile attachment strengths of different coated coupons.

FIG. 4 shows a fracture surface of HA-coated and Ag-HA-H-coated samples.

FIG. 5 shows the silver release profile in PBS of a sample.

FIGS. 6 a (HA) 6 b (Ag-HAD and 6 c (Ag-HA2), shows secondary electronimages of each different sample at low magnification, providing anoverview of each sample type.

FIGS. 7 a (HA), 7 b (Ag-HAD and 7 c (Ag-HA2) show higher magnificationsecondary electron images of the surface morphology of the samples ofFIG. 6.

FIGS. 8 a (HA), 8 b (Ag-HA1) and 8 c (Ag-HA2) show backscatteredelectron images of different samples at low magnification, providing anoverview of each sample type.

FIGS. 9 a (HA), 9 b (Ag-HA1) and 9 c (Ag-HA2) show higher magnificationbackscatter images are shown from each of the samples in FIG. 8.

FIGS. 10 a (Ag-HA1) and 10 b (Ag-HA2) and 1 Ia (Ag-HA1) and 1 Ib(Ag-HA2) show higher magnification examination of samples of FIGS. 8 and9.

FIG. 12 is a backscatter EM of a sample of the present invention.

FIG. 13 a shows an elemental dot-map obtained by EDX microanalysis,while a backscatter image of the corresponding area of sample Ag-HA2 isshown in FIG. 13 b.

FIGS. 14 a (Ag-HA1) and 14 b (Ag-HA2) shows the EDS spectra from Ag-HA1sample and Ag-HA2 sample.

FIG. 15 shows the EDX spectra of discrete bright particles on HA2samples (spots marked with “x” in images).

FIGS. 16 a-c is an image of the areas of the coating away from discretebright particles that were also evaluated via EDXA on HA2 samples.

FIG. 17 shows the X-Ray Diffraction pattern of the three samples, HA,Ag-HA1, and Ag-HA2.

FIG. 18 shows the tensile bonding strength of the three differentsamples.

FIG. 19 is a schematic of an embodiment of a coating of the presentinvention.

FIG. 20 is a low magnification of the top PLGA coating containing silvermodified beta-TCP and Bupivacaine.

FIG. 21 is a high magnification of the top PLGA coating containingsilver modified beta-TCP and Bupivacaine.

FIG. 22 is a schematic of another embodiment of the present invention.

FIG. 23 is a low magnification of the top view of the PLGA beads on aPLGA coating.

FIG. 24 is a high magnification of the top view of the PLGA beads on aPLGA coating.

FIG. 25 is a high magnification of the PLGA coating.

FIGS. 26 (a) and (b) are SEM images from 9-day pulled out implant: LowAg-modified calcium phosphate-coated implant from Rabbit # IA.

FIGS. 27 (a) and (b) are SEM images from 9-day pulled out implant:non-calcium phosphate-coated implant from Rabbit # IA.

FIGS. 28 (a) and (b) are SEM images from 9-day pulled out implant: HighAg-modified calcium phosphate-coated implant from Rabbit # IB.

FIGS. 29 (a) and (b) are SEM images from 9-day pulled out implant:non-calcium phosphate-coated implant from Rabbit # IB.

FIG. 30 is a back-scattering SEM of a ‘low’ S-CP, 9 days. (Sample 4ARight). Small regions of mineralized tissue (bone) (arrows) withinregions of the porous coat. Dashed lines shows the position of host boneafter site drilling.

FIG. 31 is a back-scattering SEM of a ‘high’ S-CP, 9 days. (Sample 5BLeft). Small regions of mineralized tissue (bone) (arrows) withinregions of the porous coat. Dashed lines shows the position of host boneafter site drilling.

FIG. 32 is a back-scattering SEM of a ‘control’ (no CP), 9 days. (Sample5B Right). Small regions of mineralized tissue (bone) (arrows) withinregions of the porous coat.

FIG. 33 is a back-scattering SEM of a ‘low’ S-CP, 16 days. (Sample 9CRight). Extensive bone ingrowth throughout full porous coat depth.Dashed lines shows initial drilled bone border.

FIG. 34 is a back-scattering SEM of a ‘high’ S-CP, 16 days. (Sample 8DRight). Extensive bone ingrowth throughout full porous coat depth.Dashed lines show probable initial drilled bone border.

FIG. 35 is a back-scattering SEM of a ‘control’ (no CP), 16 days.(Sample 2C Left). Bone ingrowth throughout depth of porous coating;difficult to identify initial drilled bone border.

FIGS. 36 (a) and (b) show a 9-day sintered porous-coated Ti6A14V‘control’ implant—(a) and (b) Sample 5B Right—the blue-green stainedareas are bone (old and newly-formed). Due to the section thickness,some bone does not show the staining effect and appears grey. A smallamount of fibrous tissue is present near the interface in some regions(arrow).

FIGS. 37 (a) and (b) show the 9-day sintered porous-coated H6A14Vimplant with ‘low’ S-CP over-layer—(a) Sample 8A Left, (b) sample 4ARight—In (b), the extent of original bone loss due to drilling (andpossibly some bone die-back) is evident by the truncated trabeculae.

FIGS. 38 (a) and (b) show the 9-day sintered porous-coated Ti6A14Vimplant with ‘High’ S-CP over-layer—(a) & (b) Sample 8B Left—Both thehigh and low magnification images show the extent of bone loss due tosite preparation (drilling) and possibly subsequent bone die-back(dashed line in (b). Nevertheless, a suitable press-fit was achievedallowing early bone formation within the interface zone and into theporous coat (arrow).

FIGS. 39 (a) and (b) show the 16-day sintered porous-coated T16A14Vimplant ‘control’ implant—(a) & (b) Sample 2C Left—Extensive new boneformation and ingrowth throughout the porous coat (blue-green stainedareas).

FIGS. 40 (a) and (b) show the 16-day sintered porous-coated Ti6A14Vimplant with ‘Low’ S-CP over-layer—(a) & (b) Sample 9C Right—Extensivenew bone formation and ingrowth. [Sample embedding artifacts (airbubbles) seen in (a)].

FIGS. 41 (a) and (b) show the 16-day sintered porous-coated H6A14Vimplant with ‘High’ S-CP over-layer—(a) & (b) Sample 8D Right—Good boneingrowth along implant length.

FIGS. 42 and 43 show results of sonicated counts for microbiologicalactivity.

FIGS. 44 and 45 show results of suspension counts for microbiologicalactivity.

FIG. 46 shows the mean absorbance at A450 nm-A655 nm of MC3T3 cellscultured in Ag/HA cultured media. Error bars represent the standarddeviations of the data.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following description of the depicted embodiment(s) is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

An antimicrobial HA powder formulation is formed by first soakingconventional hydroxyapatite (HA) powder (such as a commerciallyavailable HA powder having an average particle size of about 45 to about125 microns) in a silver nitrate-containing and/or silverfluoride-containing aqueous or organic solution for a period of time. Insome embodiments the aqueous or organic solution may comprise bothsilver fluoride and silver nitrate. In some embodiments, beta tricalciumphosphate may be used or HA may be combined with beta tricalciumphosphate. The calcium phosphate mixture includes about 0.1 percent toabout ten percent by weight of silver, from about 0.1 wt % to about 7 wt%, or from about 0.1 wt % to about 5 wt %. In one particular embodiment,the calcium phosphate mixture includes about 0.5 percent to about threepercent by weight of silver, or from about 0.5 wt % to about 2 wt %.

The term “wt %” or “weight percent” refers to the % of the weight of thecoat or the layers within the coat, and does not include the weight ofthe implant itself.

In some embodiments, one or more of carbonate, fluoride, silicon,magnesium, strontium, vanadium, lithium, copper, and zinc may be addedto the calcium phosphate mixture.

The HA powder soaks and stirs in the solution for a period of about oneday to about seven days. It is preferred that the HA powder soaks andstirs in the solution for a period of approximately one day to threedays to allow sufficient ion exchange reaction between HA powders and AgSalt(s) solution. The reaction is more preferably about two days.Further, for best results, it may be necessary to avoid excessiveexposure of the mixture to light.

The HA powder may soak at room temperature, however, a slightly warmertemperature is preferred in order to increase solubility of the solutioninto the HA. The temperature should not be elevated so much as to breakdown the composition. In some embodiments, a temperature range of about20 degrees Celsius to about 95 degrees Celsius may be used. In otherembodiments, a temperature range of about 60 degrees Celsius to about 80degrees Celsius may be used.

It is also generally important to maintain a proper pH level of thesolution during the time in which the HA powder is reacting with AgSalts in the aqueous or organic solution. The pH should generally bekept at level such that there is a minimum of HA dissolution but at thesame time reduce the possibility that the silver nitrate will react withOH and precipitate to form silver hydroxide and eventually becomessilver oxide. The pH level may be maintained in the range of 6.5-8.5,but is preferably above 6.7 to prevent the HA from dissolving. Morepreferably, the pH level of the mixture is in the range of 6.8-7.2, butother levels may be acceptable.

After the ion exchange reaction, the mixture may be left to air dry ormay be rinsed and/or washed with deionized and distilled water (DDH₂O)and then allowed to air dry.

The remaining solid mixture comprises a homogenous HA powder formulationhaving silver and/or fluoride therein. The homogenous HA powderformulation may be ground up and then used in a conventional plasmaspray process to coat a biomedical implant or medical device as wouldnormal HA powder. The soaking process of the present invention generallyonly negligibly increases the mean particle size of the HA powder, andthereby does not interfere with subsequent plasma spray processes thatmay be dependent on conventional equipment.

In a first method, HA powder is combined with an AgF solution. Themixture soaks and is stirred for one to three days. The mixture isdried. The resulting formulation may be plasma sprayed onto a medicalimplant. As an example, the silver fluoride may have a concentration ofabout 1×10″ to about 1×10″ M. As an example, the mass ratio of silverfluoride to HA powder is within the range of about 0.01 to about 0.1. Inone embodiment, the mixture is dried in air for about one day to aboutthree days at a temperature of between about 50 degrees Celsius to about95 degrees Celsius. In yet another embodiment, the silver solution has aconcentration in the range of 1×10″³ to about 1×10″⁴ M.

In a second method, HA powder is combined with an AgNO₃ solution. Themixture soaks and is stirred for one to three days. The mixture isdried. The resulting formulation may be plasma sprayed onto a medicalimplant. As an example, the silver nitrate may have a concentration ofabout 1×10⁻² to about 1×10″⁴ M. As an example, the mass ratio of silvernitrate to HA powder is within the range of about 0.01 to about 0.1. Inone embodiment, the mixture is dried in air for about one day to aboutthree days at a temperature of between about 50 degrees Celsius to about95 degrees Celsius.

In a third method, HA powder is combined with an AgF and AgNO₃ solution.The mixture soaks and is stirred for one to three days. The mixture isdried. The resulting formulation may be plasma sprayed onto a medicalimplant.

In a fourth method, HA powder is combined with an AgF solution. Themixture soaks and is stirred for one to three days. The mixture isrinsed. The mixture is then dried. The resulting formulation may beplasma sprayed onto a medical implant.

In a fifth method, HA powder is combined with an AgNO₃ solution. Themixture soaks and is stirred for one to three days. The mixture isrinsed. The mixture is then dried. The resulting formulation may beplasma sprayed onto a medical implant.

In a sixth method, HA powder is combined with an AgF and AgNCB solution.The mixture soaks and is stirred for one to three days. The mixture isrinsed. The mixture is then dried. The resulting formulation may beplasma sprayed onto a medical implant.

In some embodiments, the silver-containing HA powders are produced bysol-gel method by (a) mixing calcium, silver, and/or phosphorusprecursors to obtain a homogenous sol-gel solution; (b) aging thesol-gel solution at a temperature between about 20 degrees to about 95degrees Celsius for about seven to about 9 days; (c) drying andcalcining the sol-gel solution at an elevated temperature ranging fromabout 500 to about 800 degrees Celsius for about 2 to about 4 hours; and(d) grinding and sieving the calcined powders above to a desiredparticle size distribution for subsequent plasma spraying process. Insome embodiments, the average particle size is about 45 to about 150microns. As examples, the calcium precursor may be calcium nitrate, thesilver precursor may be silver nitrate, and the phosphorus precursor maybe ammonium dihydrogen phosphate. The silver precursor concentration maybe in the range from about 0.1 weight percent to about 10 weightpercent, from about 0.1 wt % to about 7 wt %, or from about 0.1 wt % toabout 5 wt % and more preferably from about 0.5 weight percent to aboutthree weight percent or from about 0.5 wt % to about 2 wt %.

In some embodiments, a base layer or primer layer may be applied to thesubstrate prior to application of the calcium phosphate coating. Thebase layer may be used to avoid or reduce a reaction between thesubstrate and the coating. Alternatively, the base layer may improve thetensile bonding strength at the coating-substrate interface. The baselayer may be applied using a vacuum plasma spraying process.Alternatively, the base layer may be applied using atmospheric plasmaspraying, ion sputtering, sol-gel dip coating method, solutionprecipitation, biomimetic method, or electrodeposition. The base layermay be any number of compounds. As examples, the base layer may be ametallic coating, a ceramic coating, or a biodegradable bioceramiccoating. In particular, the base layer may include calcium phosphate,bioglass, calcium polyphosphate, tetracalcium phosphate (TTCP),β-tricalcium phosphate (TCP), β-calcium pyrophosphate (CPP), β-calciummetaphosphate (CMP), or substantially pure HA. In one particularembodiment, the base layer thickness is about 1 to about 50 microns, andmore preferably about 10 to about 20 microns. In one specificembodiment, the total thickness of base layer and the antimicrobialcoating is about 30 to about 300 microns, and more preferably about 50to about 100 microns.

In some embodiments, fluorine and carbonate precursors may be mixed withthe calcium, silver, and phosphorous precursors to obtain a homogenoussol-gel solution. As examples, the fluorine precursor may be ammoniumfluoride and the carbonate precursor may be ammonium carbonate. Thefluorine precursor concentration may be in the range from about 10⁻² toabout 10″ M. The carbonate precursor concentration may be in the rangefrom about 0.1 to about 10″³ M.

In some embodiments, bone stimulating materials are mixed with thecalcium, silver, and phosphorous precursors to obtain a homogenoussol-gel solution. As examples, salts, minerals, metals, metal oxides,carbonate, fluoride, silicon, magnesium, strontium, vanadium, lithium,copper, or zinc precursors, or a combination thereof, may be mixed withthe calcium, silver, and phosphorous precursors to obtain a homogenoussol-gel solution.

One of ordinary skill in the art may readily appreciate that the exactamount or concentration of silver nitrate and/or silver fluoride in thesolution may vary. Only a small portion of the silver nitrate and/orsilver fluoride may be absorbed into the HA powder, and therefore,concentration of the solution may be optimized for reduction of silvernitrate or silver fluoride waste. Alternatively, extra silver fluorideand/or silver nitrate may be added to the solution to compensate forsome evaporation/vaporization of silver and/or fluorine during plasmaspray.

In addition, strontium and/or vanadium may be used alone or incombination with the silver and/or fluoride of the present invention, aswell as other metals like copper and zinc.

By adding a small amount of silver and/or fluoride to conventional HApowder, the present invention provides improvedantimicrobial/anti-infection/osteointegration properties to a biomedicalimplant. Moreover, the prior art fails to provide a homogeneousformulation HA powder capable of being plasma sprayed as does thepresent invention. The homogenous HA/silver powder formulation of thepresent invention may provide a much more uniform and controlleddegradation coating after plasma-spraying than the non-homogeneousapplication of silver and HA separately.

Although the examples above include hydroxyapatite, those havingordinary skill in the art would understand that other forms of calciumphosphate may be used. As examples, apatites non-stoichiometricapatites, calcium phosphates, orthophosphates, monocalcium phosphates,dicalcium phosphates, tricalcium phosphates, whitlockite, tetracalciumphosphates, amorphous calcium phosphates may be substituted for HA.

The present invention also provides a variety of medical implants,preferably coated using any of the methods and techniques as describedherein. The coating of the medical implant can comprise one or morelayers, in which each layer may comprise the same or differentcomposition as another layer. Each layer is preferably plasma sprayed ina reducing environment (e.g. in a vacuum).

Optionally, the medical implant has a coating which comprises a numberof layers, and in which the concentration of antimicrobial agent isdifferent in at least two coating layers.

The implants of the present invention are preferably manufactured toincrease the chances of successful integration into the hostenvironment, whilst at the same time providing an antimicrobialenvironment to reduce the risk of, or prevent, infection.

As various modifications could be made to the exemplary embodiments, asdescribed above with reference to the corresponding illustrations,without departing from the scope of the invention, it is intended thatall matter contained in the foregoing description and shown in theaccompanying drawings shall be interpreted as illustrative rather thanlimiting. Thus, the breadth and scope of the present invention shouldnot be limited by any of the above-described exemplary embodiments, butshould be defined only in accordance with the following claims appendedhereto and their equivalents.

EXAMPLES Example 1

METHODS

Three different concentrations of silver nitrate solutions were preparedin distilled and de-ionized water (DDH₂O) for the ion exchange reactionbetween silver nitrate solution and HA powders. Some Ag ions from thesilver nitrate solution will substitute Ca ions from the HA structure,meanwhile, some silver compounds will be physically adsorbed on the HAsurface. The target silver content in the HA powders was 0.3 wt %, 1 wt%, and 3 wt %, respectively. The mass of the silver nitrate and the HApowder was listed in Table 1.

Table 1 The mass of silver nitrate and HA powder used for the ionexchange reaction

Silver nitrate (BDH, Cat.# BDH0276-125G) was first dissolved in DDH₂Ocontained within a 3 liter glass beaker, and then the HA powders (MEDPURE®, MEDICOAT AG, particle size: −125 μm+45 μm) were added into theprepared silver nitrate solution according to Table 1. The HA and silvernitrate solution was stirred at 180 RPM in an orbital shaker at roomtemperature for 3 days to allow a homogeneous ion exchange reaction.

After the ion exchange reaction, the solution was left to dry at 80° C.in an oven to evaporate the water. The dried and modified HA powderswere then slightly ground manually using pestle and mortar to break theagglomerates formed during the drying process. The ground modified HApowders were then sieved through 100-325 mesh sieve (W.S. Tyler, Inc.,USA). Any HA powders that were larger than 150 μm or smaller than 45 μmwere discarded. At least 98% HA powders were collected between 100-325mesh sieves.

The silver modified HA powders were further characterized using a LaserDiffraction Particle Size Analyzer (Model LS 13 320, Beckman Coulter,USA). AU the measurements were listed in Table 2. The average particlesize was slightly increased with the increase of the silver content inthe HA powders. The particle size distribution of the low and mediumsilver HA powders were similar to the control pure HA powders.

Table 2 Particle size and size distribution of pure HA and modified HApowders

X-ray Diffraction (XRD) was utilized to characterize the structure,phase compositions, and the grain size of the powders (pre-spraying).This analysis was performed at H&M Analytical Services, Inc. (Allentown,N.J., USA). The coated samples were placed on a standard sample holderand put into a Philips PW3020 diffractometer using Cu radiation at 40kV/30 mA. Scans were run over the range of 10° to 70° with a step sizeof 0.02° and a counting time of 8 hours each. Scanning ElectronMicroscopy coupled with Energy Dispersive X-ray Analysis (JEOLJSM-6460LV, Japan) was used to examine the surface morphology, Ca/Pratio, Ag content of the coating, and the coating thickness.

The silver modified powder was further analyzed for composition bycompletely dissolving the powder (pre-spraying) in 10% Nitric acidsolution. An aliquot was withdrawn from the nitric acid solution forsilver ion concentration analysis using inductively coupled plasma (ICP)mass spectroscopy.

These silver modified HA powders were then used in a conventional vacuumplasma spray process at Medicoat (Magenwil, Switzerland) for the silvercontaining HA coatings. All substrates onto which the powders weresprayed were made from mill-annealed Ti6A14V alloy. The flat-surfaceddisc samples were 12.6 mm in diameter and 3.1 mm in thickness. Thesecoupons were grit blasted and cleaned prior to plasma spraying. All thespray parameters were the same for both pure HA powders and the silvermodified HA powders. A stainless steel plate with 012.6 mm holes wasused to mount Ti6A14V coupons. After the coating process, the as-sprayedcoatings were rinsed with isopropanol.

X-ray Diffraction (XRD) was utilized to characterize the structure,phase compositions, and the grain size of the coatings in the samemanner used for the powders.

Three one-inch diameter Ti6A14V coupons from each of four groups wereused as substrates to deposit HA or Ag doped HA coatings to assess thetensile attachment strength of the coatings in a manner similar to thatdescribed in ASTM F1 147.

The back-side of each coupon and the ends of the testing stubs were alllightly sanded with 80-grit paper to enhance epoxy attachment strength.The back-side of the coupons (i.e. no-coated surface) was cleaned withacetone and allowed to air-dry for 2-3 minutes.

Tensile testing stubs were glued to the opposing surfaces of each couponusing one layer of FM1000 adhesive on the back surface and one layer onthe coating surface. This was done by assembling the constructs in acuring fixture and placing the fixture in a convection oven for 130minutes (or 135 minutes if two fixtures were put in the ovensimultaneously) at a temperature of 338° F. (170° C.) under a deadweight load of 2.3 lbf (10 N) to thermal cure the glue. After removingthe fixture from the oven and allowing it to cool to room temperature,the dead weights were removed and the constructs were taken out of thefixture.

Each construct was tested by subjecting it to a tensile load at adisplacement rate of 0.10 in/min (2.5 mm/min) until failure. The peakload and failure mode for each construct was recorded. The tensileattachment strength was determined by dividing the peak load by thecross-sectional area of the coupon. After testing, each coupon wasinspected to ensure that glue did not penetrate to the substrate, whichwould indicate an invalid test.

One sample was used for each condition for silver release evaluationfrom the coating. The coated disc samples were soaked in 2 mL of PBS(pH=7.4) at 37° C. for 24, 72, and 168 hours. At each time point, ImLsolution was withdrawn and stored in an eppendorf tube at 4° C.refrigerator before Inductively Coupled Plasma (ICP) silver analysiswhich was carried out at Environmental Testing & Consulting, Inc.Memphis, Tenn. 15 μL of concentrated nitric acid was added into the tubeto prevent the silver deposition from the solution.

RESULTS

The XRD and ICP results of the silver modified HA powders are listed inTable 3 below. The Total Ag (XRD) is the result from the powder XRDphase composition analysis. XRD results demonstrated that the silver waspresent as either metallic silver, silver nitrate (i.e. the originalsilver ion exchange reaction media containing silver), or silver oxide.It should be noted that XRD only determines crystalline phases ofmetallic silver or silver compounds. It does not detect silver that hasbeen substituted into the HA crystal. ICP was used to determine thetotal amount of Ag in the silver modified HA powder. ICP analysismeasures all silver present-silver present as substituted in the HAcrystals and silver present as a discrete compound separate from the HAcrystals (e.g. silver nitrate, silver oxide, and metallic silver). Theamount of silver contained in the HA crystals can be determined bysubtracting the XRD from the ICP results. For example, for the 1 wt %Ag—HA powder, XRD results showed there was 0.45 wt % of silver in thesilver modified HA powder; however, the ICP result of the same powdershowed the total Ag content in this modified powder was 0.98 wt %, whichwas close to the designed total silver concentration. Therefore, thisindicates that about 54% of silver was substituted into the HAstructure. The excess silver from silver nitrate ion exchange solutionwas converted to other Ag phases as identified by XRD after VPS process.

Table 3

FIG. 1 represents the scanning electron micrograph of the pure HA (a),Ag-HA-L (b), Ag-HA-M (c), and Ag-HA-H (d)-coated Ti6A14V disc samplesshowing the surface appearance of the coatings. In general, all thecoatings appeared uniform and covered the Ti6A14V substrate. There wasno obvious difference between the silver containing HA coatings and thepure HA coatings. A cross section of the HA and the Ag-HA-H samplesindicates the thickness of the coating is about 80 μm, FIG. 2. Thesilver concentration was measured at different locations throughout thecoating for the Ag-HA-H sample, as indicated by the “x” markers.

Quantitative energy dispersive X-ray analysis (EDXA) shows the Ca/Pratio and Ag contents in the coatings in Table 4. At least 10-15 randomselected areas were used per sample to collect the spectra for thisquantitative analysis. The Ca/P ratio decreased with increasing silvercontent, which indicates an increasing substitution of Ag in the HAlattice with increasing Ag in the coating. Furthermore, Ag wasidentified through the thickness of the Ag-doped coatings (FIG. 2; Table5).

The Ag contents of the doped HA coatings were higher than that measuredby ICP (Table 3), which is likely due to surface roughness artifactsassociated with EDXA analysis.

Table 4

Ca/P ratio and silver content analysis using EDAX

Table 5

Ag concentration measured by EDXA across the thickness of the coating.Positions and corresponding locations are shown in FIG. 2 for thissample.

The XRD results are presented in Table 6. A quantitative analysis wasperformed using a Rietveld refinement. Besides HA phase, an impurityphase of Ca₃(PO₄̂CaO (Tetracalcium Phosphate, TTCP) was identified inall the coated samples. There was no Ag detected in the control pureHA-coated samples. The Ag content in the doped HA coatings was close tothe designed Ag content shown in Table 1. The loss or evaporation of Agduring VPS process was minimal. Moreover, the addition of Ag into the HAcoatings did not significantly alter the phase compositions for theAg-HA-L and Ag-HA-M-coated samples; however, the high Ag doped HAcoatings (i.e. Ag-HA-H) showed a slightly increased HA phase anddecreased TTCP phase than the control pure HA-coated samples.

Table 6

Phase determination and composition of VPS-coated samples QuantitativePhase Analysis: Weight Fraction (wt %) Sample Ca₁₀(PO₄)₆(OH)₂Ca₃(PO₄)₂CaO Ag

HA 83.9+0.6% 16.1+0.5% ND*

Ag-HA-L 83.9+0.8% 15.8+0.6% 0.3+0.1% Ag-HA-M 83.3+0.8% 15.6+0.5%1.1+0.2% Ag-HA-H 85.8+0.9% 11.6+0.4% 2 6+0.2% *ND: Not Detectable

In addition, the lattice parameters and the grain size of the HA phaseand the Ag phase were calculated using Scherrer's formula. These resultsare described in Table 7. The full width at half- maximum (FWHM) datawas used along with Scherrer' s formula to determine the average HA andAg grain size. Scherrer's formula is given by:

J $ cos ft where λ is the X-ray wavelength, β is the FWHM and θ is theBragg diffraction angle.

Table 7 Lattice parameters and grain size of coated samples

It is worth noting that all coated samples (including the Ag doped HAcoatings) had a significant higher c/a ratio as compared to thestoichiometric standard HA (Table 7). This indicates the HA andAg-HA-coated samples are non-stoichiometric which was evidenced by theCa/P ratios shown in Table 4.

Using Scherrefs formula, the average HA grain size in the VPS coatingsranged from 40.8 nm to 57.0 nm. There was no correlation between thegrain size and the content of Ag in the coatings. The average Ag grainsize in the coatings was also in the nano scale, ranging from 37.6 nm to56.4 nm. After VPS process, Ag or silver compounds were converted tonanocrystallined metallic Ag. This nanocrystalline HA phase and metallicAg phase resulted from the fast cooling after the melted or partialmelted HA or Ag-HA powders went through the plasma and rapidly depositedonto the substrate. Therefore, there was no time for the grain growth tooccur.

Tensile attachment strength results are reported in FIG. 3. The tensileattachment strength for the Ag-HA-H-coated samples was the lowest amongall the testing samples and did not meet the ISO requirement, whichrequires the minimum adhesion strength to be 15 MPa. The addition of Agless than or equal to 1 wt % (i.e. Ag-HA-L and Ag-HA-M) did notsignificantly affect the tensile attachment strength of the coating. AUthe failures occurred within the coating. There was no obviousdifference in the appearance of the fracture surfaces between the silvercontaining HA coatings and the pure HA coatings (pure HA and Ag-HA-Hshown in FIG. 4).

The silver release profile in PBS is shown in FIG. 5. It shows that allcoated samples released Ag into the PBS at 37° C. as early as 24 hours.The released Ag concentration in PBS increased gradually over time up to7 days. The amount of Ag released into the PBS is Ag dosage dependent.The Ag-HA-H-coated sample released the highest amount of Ag into thePBS, and the Ag-HA-L-coated sample released the lowest amount of Ag.

Example 2

Hydroxyapatite Powder Modification Using Silver Nitrate Solution

Two different concentrations of silver nitrate solutions were preparedin distilled and de-ionized water (DDH₂O) for the ion exchange reactionbetween silver nitrate solution and HA powders. In theory, some Ag ionsfrom the silver nitrate solution will substitute Ca ions from the HAstructure, meanwhile, some Ag ions will be physically adsorbed on the HAsurface. The target silver content in the HA powders was 1 wt % and 2 wt%, respectively. The mass of the silver nitrate and the HA powder waslisted in Table 8.

Table 8 The mass of silver nitrate and HA powder used for the ionexchange reaction

Silver nitrate (BDH, Cat.# BDH0276-125G) was first dissolved in DDH₂Ocontained within a 3 liter glass beaker, and then the HA powders(MEDPURE®, MEDICOAT AG, particle size: −130 μm+45 μm) were added intothe prepared silver nitrate solution according to Table 8. The HA andsilver nitrate solution was stirred at 180 RPM in an orbital shaker atroom temperature for 3 days to allow a homogeneous ion exchangereaction.

After the ion exchange reaction, the solution was left to dry at 80° C.in an oven to evaporate the water. The dried and modified HA powderswere then slightly ground using an automated pestle and mortar (Retsch,Mortar Grinder RM200, Newtown, Pa.) to break the agglomerates formedduring the drying process. The ground modified HA powders were thensieved through 100-325 mesh sieve (W.S. Tyler, Inc., USA). Any HApowders that were larger than 150 μm or smaller than 45 μm werediscarded. At least 98% HA powders were collected between 100-325 meshsieves.

The silver modified HA powders were further characterized using a LaserDiffraction Particle Size Analyzer (Model LS 13 320, Beckman Coulter,USA). AU the measurements were listed in Table 9. The average particlesize was slightly increased with the increase of the silver content inthe HA powders. The particle size distribution of the low and mediumsilver HA powders were similar to the control pure HA powders.

Table 9 Particle size and size distribution of pure HA and modified HApowders

These silver modified HA powders were then used in a conventional vacuumplasma spray process at Medicoat (Magenwil, Switzerland) for the silvercontaining HA coatings.

Substrate Preparation

All substrates were made from mill-annealed Ti6A14V alloy. Theflat-surfaced disc samples were 12.6 mm in diameter and 3.1 mm inthickness. These coupons were grit blasted and cleaned at S&N in Aarau,Switzerland.

Coating Deposition

The coating was applied using the conventional vacuum plasma spraysystem at Medicoat in Magenwil, Switzerland. All the spray parameterswere the same for both pure HA powders and the silver modified HApowders. A stainless steel plate with Φ 12.6 mm holes was used to mountTi6A14V coupons. After the coating process, the as-sprayed coatings wererinsed with isopropanol.

Coating Characterization Techniques

Field Emission Scanning Electron Microscopy

The samples were examined using a FEI Nova NanoSEM 200 Scanning ElectronMicroscope (SEM) equipped with a dual backscatter detector (Dual BSD)and an EDAX Genesis 4000 Energy Dispersive X-ray microanalysis (EDX)system according to SOP/MS/131. All three examples of each sample couponwere mounted on a 25 mm diameter aluminium pin stub by means of selfadhesive carbon rich discs. The samples were examined uncoated (nosputter coating) using low vacuum conditions (0.6-1.0 Torr, watervapour) at a moderate accelerating voltage of 10 KV to minimize surfacecharging and to maximise surface information (minimised electron beampenetration).

The analytical conditions used to obtain EDX data were a 10 KVaccelerating voltage, spot size 5.5, with the final aperture in position3 and X-ray count rates of between approximately 1500 and 4000 countsper second at 20% to 39% detection system dead-time (approximatelyoptimal conditions). EDX microanalysis was carried out at 10 KVaccelerating voltage, because this provides the minimum electron beamenergy required to excite x-rays from the chemical elements of interestto this study, whilst attempting to minimise penetration of the samplepast the surface of the material. High resolution imaging was achievedwith a Low Vacuum Detector (LVD) for secondary electrons and a GaseousAnalytical Detector (GAD) for backscattered electrons. Digital images ofrepresentative areas were recorded in uncompressed TIFF format. ImagePro Plus software was used to estimate the size of silver rich materialin samples HA1-Ag/HA (TO035B) and HA2-Ag/HA (TO035C), using the SEMimage scale bar to provide a spatial calibration.

X-ray Diffraction

X-ray Diffraction (XRD) was utilized to characterize the structure andphase compositions. This analysis was performed at Swiss FederalLaboratories for Materials Testing and Research in Switzerland. The XRDpatterns were measured between 20° and 60° at a step size of 0.02° witha copper x-ray source. The signal was measured for 3 seconds at eachstep and the intensity is given in counts per second (cps). Thefollowing slits and filters were used in the following order 1 mm, 0.5mm, Ni filter, and 0.2 mm. The XRD was measured on the as sprayedcoating rather than the powder scrapped from the surface (as stated inthe ISO 13779-3). The XRD patterns of the hydroxyapatite coatings wereanalysed by a modified version of the ASTM F 2024-00 and the ISO 13779-3standards. The direct application of both standard test methods were notapplicable because of the presents of the silver obscured the results.The reasons for the modification of the standards and the methods usedto analysis the coatings were discussed in the results section. Thereference materials were hydroxyapatite (HAref76/800/7h), Al₂O₃ (AlphaAesar 99.99%) and a silver plate (99.95%).

Tensile Attachment Strength of Coatings to Ti6A14V Substrate

Five one-inch diameter Ti6A14V coupons from each of three groups wereused as substrates to deposit HA or Ag doped HA coatings to assess thetensile attachment strength of the coatings in a manner similar to thatdescribed in ASTM F1 147.

The back-side of each coupon and the ends of the testing stubs were alllightly sanded with 80-grit paper to enhance epoxy attachment strength.The back-side of the coupons (i.e. no-coated surface) was cleaned withacetone and allowed to air-dry for 2-3 minutes.

Tensile testing stubs were glued to the opposing surfaces of each couponusing one layer of FM1000 adhesive on the back surface and one layer onthe coating surface. This was done by assembling the constructs in acuring fixture and placing the fixture in a convection oven for 130minutes (or 135 minutes if two fixtures were put in the ovensimultaneously) at a temperature of 338° F. (170° C.) under a deadweight load of 2.3 lbf (10 N) to thermal cure the glue. After removingthe fixture from the oven and allowing it to cool to room temperature,the dead weights were removed and the constructs were taken out of thefixture.

Each construct was tested by subjecting it to a tensile load at adisplacement rate of 0.10 in/min (2.5 mm/min) until failure. The peakload and failure mode for each construct was recorded. The tensileattachment strength was determined by dividing the peak load by thecross-sectional area of the coupon. After testing, each coupon wasinspected to ensure that glue did not penetrate to the substrate, whichwould indicate an invalid test.

RESULTS AND DISCUSSION

SEM/EDX

FIG. 6 a to FIG. 6 c, shows secondary electron images of each differentsample at low magnification, providing an overview of each sample type.No qualitative differences can be seen in the surface appearance of eachsample type. The surfaces had a mixture of rounded, amorphousmorphologies, with some areas of aggregated, angular particulatessuggestive of incomplete melting of the powder stock used in the coatingprocess. This is consistent with the use of a melt sprayed depositionprocess such as vacuum plasma deposition, to coat the metal coupons.

FIG. 7 a to FIG. 7 c show higher magnification secondary electron imagesof the surface morphology of each sample. A mixture of morphologies canbe seen on each sample, with both amorphous-like material and some fine,angular particulates being present. Microscopic cracks were present onthe surface of the HA layer (evident in FIG. 7 c). Similar particulatematerial was observed on all of the samples and there were no apparentqualitative differences in their surface appearance regardless of sampletype or magnification, although backscattered electron signals in bothsecondary and backscatter detector images revealed the additionalpresence of micro and nano-scale silver particles in samples Ag-HA1 andAg-HA2.

FIG. 8 a to FIG. 8 c, show backscattered electron images of eachdifferent sample at low magnification, providing an overview of eachsample type. Several bright speckles/particles can be seen on thesilver-containing samples. More electrons are backscattered frommaterials composed of higher average Atomic Number (Z), creating brightareas on backscattered electron images. The brighter and very brightareas in FIGS. 8 b and 8 c correspond to the locations of material richin silver (silver compounds have a higher average Z than calcium HA,represented as darker areas in backscattered electron images). Highermagnification backscatter images are shown from each sample in FIGS. 9 ato 9 c, revealing fine detail in the spatial distribution of silver-richmaterial in the silver-containing samples.

The composition of the HA Control sample is homogeneous as visualised bybackscattered electrons (FIG. 9 a). Samples Ag-HAl and Ag-HA2 containingsilver are shown to be heterogeneous in terms of composition in FIGS. 9b and 9 c. A range of different sizes of silver-rich material can beseen, with more silver-rich material present in sample Ag-HA2. Imageanalysis estimates of the sizes of the silver-rich material rangedbetween approximately 15 nm and 10 μm for the silver-containing samples.Higher magnification examination highlights the different shapes of thesilver-based particulates, FIGS. 10 a and 10 b and FIGS. 11 a and 11 b.Light grey areas on each sample (such as circled in orange in FIG. 9 cand clearly visible in FIG. 12) are shown to be composed of manynanoscale silver particulates dispersed at or under the surface of thecalcium HA when viewed at high magnification.

FIG. 13 a shows an elemental dot-map obtained by EDX microanalysis,while a backscatter image of the corresponding area of sample Ag-HA2 isshown in FIG. 13 b. The dot-map provides confirmation that the brightfeatures and the light grey features shown in backscatter images of thesilver doped sample correspond to silver-rich material (blue areas ofEDX map).

The FIG. 14 a-b shows the EDS spectra from Ag-HA1 sample and Ag-HA2sample. The arrow indicates silver peak energies. It showed Ag-HA2 hadhigher Ag content in the coating.

The FIG. 15 shows the EDX spectra of discrete bright particles on HA2samples (spots marked with “x” in images). The silver concentrations inthese areas were much higher than that of areas without discrete brightparticles, as shown in the below images. These bright regions and theattendant EDXA spectra confirm the presence of metallic particles,previously discovered via XRD analyses.

The areas of the coating away from discrete bright particles were alsoevaluated via EDXA on HA2 samples, as shown in FIG. 16 a-c. These areas,devoid of bright discrete particles, also showed presence of silver,albeit at a much lower concentration. This confirms the presence ofsilver substitution within the HA matrix, as was demonstrated previouslyby XRD and ICP analyses.

X-ray Diffraction

Qualitative Analysis.

The XRD patterns for the three samples are given in FIG. 17. Sample HAshows all the expected peaks for hydroxyapatite. The Ag-HA1 and Ag-HA2samples show the same HA peaks but with the addition of a peak at 38.15°(arrow indicating the peak in FIG. 17) that is consistent with metallicsilver. A broad peak is observed at 31.1°. This peak could be attributedto the amorphous phase or alpha Tricalcium Phosphate (α-TCP) or betaTricalcium (β-TCP). The peak at 38.15° is the diffraction from silver(Ag) and the insert shows a zoomed in image of this peak.

3,3,2 Crystallinity Analysis

The crystallinity was measured by both the ASTM F 2024-00 and ISO13779-3 standards. The results for both methods are presented in Table10. The ASTM F 2024-00 measures the relative intensity of the peaksbetween 38.5° and 59° compared to an external standard (CC-AI₂O₃). Thesepeaks are not convoluted by the common impurities found inhydroxyapatite, but the relative intensities of these peaks are lowerthen the major peaks and therefore this method is less sensitive. TheISO 13779-3 standard measures relative intensity of the 10 most intensepeaks compared to a 100% hydroxyapatite reference. These measurementswere conducted on the ‘as sprayed’ samples, opposed to the powderscraped from the surface (as specified in ISO 13779-3).

3.3.3 Silver Analysis

The silver content of the two samples Ag-HA1 and Ag-HA2 were measured byrelative intensity compared to a pure silver sample. The results for thesilver content are given in Table 10. The Ag-HA1 samples have a silvercontent of 2.0+0.5% and the Ag-HA2 samples have a silver content of2.5+0.5%.

Table 10. The Crystallinity, β-TCP, and Silver Content

3.5 Tensile Attachment Strength of Coatings to Ti6A14V Substrate [00204]Tensile attachment strength results are reported in FIG. 18. The tensileattachment strength for the Ag-HA1-coated samples was the lowest amongall the testing samples, but even the lowest value of this group stillmeets the ISO requirement, which requires the minimum adhesion strengthto be 15 MPa. All the failures occurred within the coating. There was noobvious difference in the appearance of the fracture surfaces betweenthe silver containing HA coatings and the pure HA coatings.

Example 3

Gradient Coatings for Biomedical Applications

FIG. 19 shows an example of an embodiment of the present invention (animplant substrate (1510) [e.g. Ti6A14V] with a gradient coatingcontaining VPS HA (1512) and VPS AgHA (1514). and a top layer of PLGAcoating contains β-TCP, Ag, and Bupivacaine (1516)) prepared by thefollowing method:

1. HA/Ag-HA coating preparation: The Ag-HA powders (45-125 μm) weremodified using an ion exchange reaction. The coating process parameterswere the same as the standard vacuum plasma sprayed HA coatings producedat our manufacturing facility for medical implants. VPS HA coating wasfirst applied and then followed by the VPS Ag-HA coating. The coatedsample was ready for the PLGA coating.

2. Silver modified β-TCP powder preparation:

1). 0.5 g β-TCP powder (Dso-3 μm) and 145.8 mg silver nitrate weredissolved into 55 mL de-ionized and distilled water and stirred for 1hour at 60° C.

2). The water was evaporated overnight at 60° C.

3). The dry powder was then ground. Alternatively, the silver modifiedβ-TCP can also be freeze dried to remove the water and the grinding stepis not necessary.

4). The silver modified powder was subsequently sintered at 400° C. for2 hours.

3. PLGA solution preparation:

1). 0.75 g PLGA pellets (85:15) were dissolved in 15 mL ofdichloromethane and stirred overnight.

2). The 0.25 g silver modified β-TCP and 100 mg Bupivacaine powder weredissolved into the PLGA solution and stirred overnight.Asdasd

4. PLGA coating application: The VPS HA/VPS Ag-HA coated H6A14Vsubstrate was dipped into the PLGA solution and withdrawn vertically andthen dry in air overnight.

Results: The surface morphology of the top PLGA layer is shown in FIGS.20 and 21. A quantitative analysis obtained from an EDXA spectrum isshown in Table 11.

Table 11. EDXA result of the top PLGA coating

Example 4

FIG. 22 shows another embodiment of the present invention (an implantsubstrate (1810) [e.g. Ti6A14V] with a gradient coating containing VPSHA (1812) and VPS AgHA (1814), and a layer of PLGA coating containsβ-TCP, Ag, and Bupivacaine (1816), and a PLGA beads layer containingβ-TCP, Ag, and Bupivacaine (1818)).

This is an example to demonstrate that the amount and release durationof Ag and Bupivacaine can be controlled by increasing the total coatingsurface area through adding PLGA beads on the top surface of the PLGAcoating. Bupivacaine was known to have a quick release profile in thebody environment, hi order to have a continuous prolonged release,Bupivacaine was incorporated into the PLGA beads to slow down itsdegradation rate in the body enviornment.

Method

1. PLGA beads preparation:

1) The silver modified β-TCP powder was prepared in the same way as inthe Example 3.

2) The 0.25 g silver modified β-TCP and 100 mg Bupivacaine powder weredissolved into the PLGA solution (0.75 g PLGA in 15 mL dichloromethane)and stirred overnight.

3) 5 g Sodium Dodecyl Sulfate (SDS) was dissolved into 500 mL de-ionizedand distilled water.

4) The PLGA solution containing the silver modified β-TCP andBupivacaine powder was added into the SDS solution drop by drop with avigorous stir. The beads formed from the water-oil-water doubleemulsification were washed and collected after 24 hours stirring in the1% SDS.

5) The collected PLGA beads were applied onto the top PLGA coating whichalso contains silver modified β-TCP and Bupivacaine.

6) The PLGA beads were sintered together and to the PLGA coating at 70°C. for 12 hours.

Results: The surface morphology is shown in FIGS. 23, 24, and 25. Thesurface composition was analyzed using EDXA and the result was shown inTable 12.

Table 12: EDXA result of the top PLGA coating

Example 5

Release Profiles

The release of Ag, Ca, and Bupivacaine from the prepared coating wasconfirmed by ICP analysis and UV spectrometry, respectively.

The coated samples were immersed in 3 mL PBS for 24 and 48 hours at 37°C. At each time point, the release of bupivacaine was measuredspectrophotometrically (Nanodrop, Thermo) at 265 nm. The Bupivacainestandard was prepared by dissolving appropriate amounts of the drug inPBS. PBS was used as blank. The Bupivacaine concentration is shown inTable 13.

Table 13: The Bupivacaine concentration in PBS at 24 and 48 hours

The Ag, Ca. and P concentration in PBS at 24 and 48 hours were shown inTable 14.

Table 14: The silver and calcium concentration (ppm) in PBS at 24 and 48hours

The degradation study confirmed the coating was able to releaseBupivacaine for analgesic effect, Ag ions for antimicrobial effect, andCa for osteoconductive effect.

Example 6

Example of method of synthesis and characterization

Synthesis

1). VPS gradient coating: Ti6A14V substrate+pure VPS HA layer+3% VPSAgHA layer

2). Dissolve PLGA (85:15) pellets in dichloromethane and stir overnight3). Soak and stir β-TCP in silver nitrate solution for 2 hours to allowion-exchange reaction

3). Add Bupivacaine into the above TCP+silver nitrate solution 4). Drythe Bupivacaine+TCP+silver nitrate solution overnight 5). Add the drypowder of Bupivacaine+TCP+silver nitrate to the dissolved PLGA solutionand stir overnight

6). Dip coat the VPS gradient coating using the above PLGA solution withBupivacaine+TCP+silver nitrate

Characterization

1). SEM top view and cross section

2). EDAX - elemental composition of top layer and cross section (Ca, P,Ag) 3). XRD-Phase composition of the top layer (mainly to detectbupivacaine)

4). Alternative to XRD: dissolution of top layer in PBS (3 days) andsubsequent spectroscopic analysis of bupivacaine

Example 7

Further example of method of synthesis and characterization

Synthesis

1). Sol-gel dip coating process to make a Ag graded coating, i.e.Ti6A14V substrate+pure Ca-P layer+2% Ag-Ca-P layer 2). Dissolve PLGA(85:15) pellets in Chloroform

3). Add analgesic (e.g. over counter Tylenol) and Ag-CaP (2 wt % Ag)powders into the PLGA 4). Dip coat the sol-gel Ag-Ca-P sample using theprepared PLGA polymer solution.

Characterization

1). SEM top view before and after degradation in PBS and SBF 2).ToF-SIMS to obtain depth information 3). XRD-Phase composition 4). Invitro bioactivity evaluation in SBF (3 days) 5). Dissolution in PBS (24h, 48 h, 72 h) to measure Ag concentration.

Example 8

Osseointegration of porous-surfaced implants with modifiedanti-microbial calcium phosphate coatings.

The results of mechanical pull-out testing of Ti6A14V alloyporous-surfaced implants prepared with or without sol-gel-formedAg-modified calcium phosphate thin film overlayers (approximately 1micron thick) are reported herein. Briefly summarizing, the study used 4groups of 10 rabbits that had porous- surfaced implants (Endopore®dental implants acquired from Innova-Sybron Dental Products) implantedtransversely in their medial femoral condyles, (porous regioninterfacing with cancellous bone). Implant positioning and implantationprocedures were similar to those described in Tache et al (2004), Int JOral Maxillofac Implants, 19:19-29; Gan et al (2004), Part II:Short-term in vivo studies, Biomaterials. 25:5313-5321; Simmons et al(1999), J Biomed Mater Res., 47: 127-138, all of which are hereinincorporated by reference. ‘Test’ implants (one per animal in either theright or left leg—random placement) were prepared with Ag-modifiedcalcium phosphate coatings overlaying the sintered porous surface of theTi alloy implants. The porous surface region consisted of approximatelythree layers of Ti6A14V alloy powders (44 to 150 micron particle size)sintered so as to form a porous layer approximately 300 micron thickwith 35 volume percent porosity (approximate) and with average pore sizein the 75 to 100 microns range. The interconnected open-pored structurewas suitable for achieving implant fixation by uninhibited boneingrowth. It is noteworthy that this particle and pore size is somewhatsmaller than that conventionally used with orthopedic implants but hasproved acceptable and, in fact, is preferred for dental implantapplications where dimensional constraints arise.

The sol-gel-formed calcium phosphate overlayer had been studiedpreviously (but minus the Ag+ modification) and, in the unmodified form,was observed to promote faster bone ingrowth (i.e. enhancedosseointegration). Based on these earlier studies, Ag-modified calciumphosphate coatings were proposed and developed by Smith & Nephew asantimicrobial and osteoconductive coatings that would both increase boneingrowth into porous-surfaced implants as well as reduce the possibilityof infection at an implant site during the early post-implantationperiod. This increased infection resistance during the crucial earlypost-implantation healing period is desirable since microbial ingressresulting in local infection and inflammatory response would inhibitbone ingrowth and potentially result in implant failure. Therefore,reducing the probability of bacterial infections during this earlyperiod would be of considerable benefit in improving the reliability oforthopaedic implants designed for fixation through bone ingrowth.

Materials & Methods

Two different Ag+-containing calcium phosphate formulations wereinvestigated. These are designated in this report as ‘Low’ and ‘High’ Aglevels. (In the results presented below LC=low Ag+(0.9 wt %) calciumphosphate and HC=high Ag+(2.5 wt %) calcium phosphate coatings). Theanimal study was designed such that the LC implants were placed infemoral condyles of 20 rabbits with ‘control’ implants (i.e. no calciumphosphate (NC) sol-gel coating) in the other femur while the HC implantswere placed similarly against ‘control’ implants in the remaining 20rabbits. Ten rabbits from each group were maintained for 9 daysfollowing implant placement and then euthanized while another tenrabbits were maintained for 16 days prior to sacrifice. This provided 10LC implants after 9-day implantation for comparison against 10 NC 9-dayimplants and a similar number of LC implants for comparison with NCimplants at 16 days. Similarly two groups of 10 HC implants were studiedafter 9- and 16-day implant residence periods and compared with NCimplants.

Implant performance in terms of effective bone ingrowth leading tosecure implant fixation was assessed by mechanical pull-out testing (asin the previously reported studies as discussed above) as well ashistological examination and assessment of some of the implant-tissuesamples after animal sacrifice. Additionally, some of the pulled outimplants were examined by secondary electron imaging in the scanningelectron microscope to characterize the implant-tissue interface regionand to identify any bone-like or fibrous tissue features that might bepresent. The virtue of the mechanical pull-out testing is that this testprovides information on the complete interface rather than the selectedarea that is observed through microscopic examination. All specimens formechanical testing were stored in saline solution following animaleuthanization and dissection of the femoral condyle region and testedwithin 2 hours of sacrifice.

Eight of the 10 samples per group as described above were mechanicallytested with the remaining two specimens being used for histologicalsample preparation. Pull-out testing involved mounting the bone-implantsamples in a custom-made fixture that ensured proper alignment of theimplant and applying a pull-out force under displacement control at arate of lmm/min. The tapered shape of the porous-surfaced implant andthe careful sample alignment ensured that frictional forces acting atthe bone-implant junction that might have contributed to measuredpull-out force and interface stiffness were avoided. Maximum pull-outforce and maximum tangential slope of the load-displacement curve wereused to determine pull- out resistance and the interface zone stiffness.

Two of the 10 samples per group as described above were collected afterrabbit sacrifice and fixed in 10% buffered formalin and processed forembedding in methyl methacrylate. The resulting blocks were sectionedusing a diamond wafering blade to produce sections approximately 200micrometers in thickness along the long axis of the implants at theirmid-plane. These samples were then mounted on glass slides and carefullyground and polished to provide non-decalcified sections approximately 30to 40 microns in thickness. The ‘thin’ sections were stained with a 1:1mixture of 0.3% Toluidine blue and 2% sodium borate at 50° C. for 15minutes, and then stained in 0.3% light green in 2% acetic acid at roomtemperature for 3 minutes. The sections were examined by lightmicroscopy and appearance recorded as described below.

Statistical analyses (Analysis of Variance with implant design as theone variable parameter) of the maximum pull-out force and measuredinterface zone stiffness values for the calcium phosphate coated ‘test’implants versus the non-coated ‘control’ implants for the differentpairs of implants were undertaken. Thus, the 9-day LC implants werecompared with the corresponding 9-day NC implants placed in thecontralateral rabbit femoral condyle, the 9-day HC implants werecompared with the corresponding 9-day NC implants and the 16-day pairedimplants were compared in the same way. In addition, the 9-day NCimplants were compared with the 16-day NC implants and the 9-day HCimplants and 16-day HC implants were compared similarly.

Results & Discussion

The mechanical test results from the current study are presented inTable 15

Table 15 Summary of Mechanical Pull-out Tests

Sample Type Implant Period Interface Stiffness Pull-out Force (days)(N/mm) (Mean+(N) (Mean+SD) SD) Low Ag-CP 9 311+140 192+116 f High Ag-CP9 355+158 193+69 # Control-No CP 9 307+99*177+66 $ Low Ag-CP 16 355+89402+118 f High Ag-CP 16 432+75 413+147 # Control-No CP 16371+75*469±120φ * Significant Difference (p=0.048) f, $, # SignificantDifference between pairs (p<0.01)

The statistical tests indicated that there were no significantdifferences for both maximum pull-out force and interface stiffnessbetween the ‘test’ and ‘control’ implants for all pairs of samples(significant differences corresponding to p<0.05). However, there was ahighly significant increase in pull-out force for the 16-day implantscompared with the 9-day samples for both the LC and HC implants(p<0.01). The interface zone stiffness also showed an increase from 9days to 16 days and while this increase was significant (p=0.048). thedifference was not nearly as great as that observed for pull-outresistance. This interesting result suggests that the interface zonedevelops a stronger resistance to crack propagation and fracture as moreextensive tissue and bone ingrowth develops (i.e. a ‘tougher’ interfacezone develops) from 9 to 16 days. The increase from the 9- to 16-dayimplantation period is consistent with previously reported results withthis rabbit femoral condyle implantation model.

Interestingly, the Ag+-modified calcium phosphate overlayer resulted ininterface stiffness values after the 16-day implantation period that,while higher on average than the values for the 9-day implants, were notsignificantly different. The resistance to implant removal by 9 days forboth the as-sintered, non-calcium phosphate-coated and the Ag+-modifiedcalcium phosphate coating (Low and High Ag+) indicated that tissue(bone) ingrowth had occurred for the coated implants.

SEM Examination of Pulled Out Implants

Some of the 9-day implants that had been mechanically tested wereexamined by secondary electron emission scanning microscopy.

FIGS. 26 through 29 show eight of the collected images. FIGS. 26 a&bshow images of a calcium phosphate-coated, lower Ag+ implant (CL-9)extracted from the 9-day implanted rabbit #1 A. While this implantexhibited lower interface stiffness and pull-out force, the secondaryelectron images nevertheless show extensive tissue attachment andingrowth with areas displaying the characteristics of mineralizedtissue. FIGS. 27 a&b are images of the noncoated ‘control’ implant(NCL-9) extracted from the other knee of the same animal. This implantdisplayed higher stiffness and pull-out values compared to the coatedimplant (CL) from the contralateral limb and showed the expectedextensive tissue attachment and mineralized tissue ingrowth by the 9-dayimplant period. FIGS. 28 a&b and 29 a&b are images of the extractedhigher Ag+-containing calcium phosphate coated implant (FIGS. 28 a&b)and the corresponding non-coated ‘control’ implant (FIGS. 29 a&b);(Rabbit #1B i.e. containing implants CH-9 and NCH-9 respectively).

BS-SEM Examination of Non-mechanical Testing Implants

BS-SEM was used to collect images of the tissue-implant interface zonewith quantitative image analyses being performed on the examinedsections. For the quantitative assessment (Quantimet Image Analysisprogram), an envelope approximately 220 micrometers wide from theimplant substrate along the length of its porous-coated region wasselected (i.e. an envelope width that approached the extremity of theporous coat along the implant length but excluded more peripheralregions; the implant ends were also excluded), This region was analyzedusing the Quantimet image analysis software. The percent area of bonewithin the pores was determined (i.e. % [bone area/pore area]). Theprogram also allowed a determination of the percent porosity of theporous coat that was nominally designed to be 35 to 40 volume percent.

FIGS. 30 to 35 show typical BS-SEM images for all sample types. TheBS-SEM images clearly show mineralized tissue (bone) ingrowth (lightgrey regions) at the two time periods for implants with CP over-layersas well as ‘control’ implants. The results of the quantitative imageanalysis for percent bone within available porosity are presented inTable 16. For the sections analyzed, the implant length was divided intofour sections for analysis thereby allowing higher magnification imagesfor the analysis. The four measurements were then averaged to give apercent bone ingrowth (and percent porosity) for each implant. The datafrom all the sections is included in Table 16 and indicates thevariation that was observed along the implant length. This is notsurprising in view of the structure of the cancellous bone into whichthe implants were placed. For each implant, a mean and standarddeviation was determined. A one-way ANOVA was undertaken to determine ifthere were statistical differences between implants in the contralaterallimbs for each rabbit. Statistical significance was considered atp<0.05. The different regions (bone, Ti alloy particles and unfilledpores, or at least not filled with bone) were readily distinguished bythe Quantimet imaging software allowing an objective determination ofthe percent bone fill within the available pores. Only intra-animalcomparisons were made (i.e. left and right legs within each animal).This provided seven sets for comparison including all the differentconditions (Low S-CP, High S-CP, control at 9 days and 16 days) with twoanimals assessed for each condition with one exception. Unfortunately,the one lost implant (Rabbit 2C) could not be included.

Table 16 Summary of quantitative image analysis of BS-SEM examination

9-day implants 4AL- 4AR- SAR- 5BR- 5BL- SBR- SBL- % bojw/pores &dc Ml McBAl-Mi 9dc Mh 9dc Mn 3347 1393 14 50 1739 2281 1642 942 12 77 1845 1202878 2708 2759 35 50 724 15 10 3230 2357 948 1637 so m 35 13 8 18 23 572785 3056 13.75 2687 36.42 33.41 10.20 25 16 mean 27.97 20.02 11.6321.93 28.37 31.37 8.7S 19.15 SD δ.82 8.66 2.91 5.S5 5.7δ 10.07 1.31 6.13ANOVA- pI 5.20 II 0.02 III 0,74 II0,82 I

16-day implants 9CL- 9CR- 2CL- 10L- 1DR- 80L- 8DR- 1Sdc 16dl 16dc 1Sdc16dh 16dc 16dh 4444 2681 3934 41 53 26 28 34 Q7 4321 53 18 48 12 4677 3168 33 39 5208 5303 4244 5070 50 53 42.12 38 59 4203 5306 3045 2956 53474449 43 15 4651 55 14 mean 42.63 38.75 47.53 39J6 35.35 43.82 52.36 SD9.36 12.42 S.11 5.66 7.25 7.36 2.94 ANOVA- pI 6.64 II 0.38 1 10.D751

Despite the small number of samples analyzed, the quantitative imageanalysis does suggest some interesting additional findings.

The 9-day data indicates that in two rabbits (8A and 8B), % boneingrowth was significantly higher for the S-CP-modified implants (8A,low S-CP and 8B high S-CP) compared to their respective ‘control’implants (no CP over-layer). The other two 9-day rabbits that wereanalyzed did not show significant differences.

There were no significant differences in bone ingrowth between theCP-modified and ‘control’ implants at 16 days.

As before, these findings indicate that the S-CP-over-layers do notinhibit bone ingrowth. In fact, the BS-SEM images and the quantitativeimage analysis suggest that the addition of the S-CP over-layer maypromote faster rates of bone ingrowth.

Quantitative image analysis was also used to confirm the percentporosity of the implants. The percent porosity as determined using theQuantimet software for the 17 sections analyzed was equal to 43.1±2.7%.

Histological Assessment of Rabbit Implants

The sections examined were prepared from 16 tissue-implant blocksharvested from 8 rabbits selected from the 40 rabbits and used in thestudy. Of these 16 samples for histology section preparation, theimplant was not present in one block. That implant (sample 2C, 16-day‘Low’ Ag+), presumably, had not osseointegrated but had migrated fromthe implant site after placement. The remaining 64 implants weremechanically tested (pull-out tests) to determine the shear strength andinterface stiffness of the implant-bone interface zone as discussedabove.

There was no obvious difference between implants that have been treatedeither high or low and controls (non-treated). Maturation of boneingrowth over time was the same in all animals. No reaction was observedto implants that have been treated and no obvious cell death insurrounding bone. FIGS. 36 to 41 show representative micrographs of eachcondition indicating regions of bone ingrowth for all implants. Thisfinding is consistent with the mechanical pull-out test results reportedabove.

Summary & Conclusions

1. The pull-out test results and the SEM images of the pulled outimplants confirm that tissue ingrowth resulting in secure implantfixation occurs by 9 days for porous-surfaced implants with an overlayerof Ag+-modified calcium phosphate sol-gel-formed coatings.

s2. The pull-out tests suggest that the modified coatings with the loweror higher Ag+-additions perform similarly.

3. As expected, the pull-out force for implant removal increased withincreasing implantation period with significantly higher pull-out forcesbeing recorded for the 16-day implanted samples compared with the 9-daysamples. However, the interface zone stiffness values were notsignificantly different for the 9- and 16-day implanted samples althoughthe mean values were higher for the 16-day implants.

4. While the recorded pull-out forces for the modified calciumphosphate-coated implants from the present study were not significantlydifferent from those reported in a previous study (Tache et al),significantly higher interface zone stiffness values were_observed. Thehigher interface stiffness may have been due to the longer implants_usedin the previous study (9 mm versus 7 mm length).

5. The addition of Ag+ to a sol-gel calcium phosphate film depositedover a porous-coated Ti-6A14V implant does not inhibit bone ingrowth.The two concentrations of silver that were tested appeared to givesimilar results.

Example 9

Antimicrobial activity of HA-Ag coatings r002631 Method

The coatings from Example 2 were evaluated for antimicrobial activity.In addition, a negative control (uncoated Ti6A14V substrate) and apositive control (Acticoat 7 (Smith and Nephew)—a nanocrystalline silvercontaining wound dressing known to have antimicrobial properties) werealso evaluated.

Sample Culture Method

A suspension of the test organism containing approximately 10⁴ cfu/mlwas prepared, by harvesting an overnight slope culture. The test couponswere tested on a method based on ASTM standard E2149-01 (Standard TestMethod for Determining the Antimicrobial Activity of ImmobilizedAntimicrobial Agents Under Dynamic Contact Conditions).

Samples were placed into a 24 well sterile tissue culture plate flatbottom with low evaporation lid, four of each sample type were preparedfor each time point tested. In addition a positive control sample ofActicoat 7 and negative culture control were set up (3 replicates eachper time point). Each sample was inoculated with 2 ml of test organismsuspension and plates were sealed with parafilm to minimise cultureevaporation. The samples were incubated at 37° C. with agitation at 150rpm for the relevant time period, sonicated counts were taken at 24, 48and 72 hours. Sonicated counts were not suitable for the positive andnegative method control samples. Time 0 samples were not taken forsonicated counts as without time for colonisation to occur they wouldnot have yielded any relevant information.

For additional supporting information, showing any activity displayedaway from the surface, counts were taken from the test inoculum at 0,24, 48 and 72 hours to calculate the log reduction in count in thesuspension. 3 replicates were sampled at all time points as this was alltime constraints would allow.

Sonicated Counts

Sonication in a detergent solution is a recognised method of removingattached cells from metal surfaces to assess their numbers. Here it wasused to determine if different levels of bacterial growth were seen onsamples with silver-containing HA coating and non silver-containing HAcontrols.

Coupons were washed in PBS then the excess was aspirated off, thisprocess was repeated 5 further times to give 6 washes in all. Thecoupons were placed in 9 ml of STS (0.85% salt & 1% Tween 20 & 0.4%sodium thioglycolate) in 15 ml Falcon tubes and floated in a sonicatingwater bath using a polystyrene float. Samples were then sonicated for 10minutes at 60 Hz.

The resulting sonicate was diluted down to 10″⁵ in Maximum RecoveryDiluent (MRD) and all dilutions were plated out on duplicate AerobicCount Petrifilm (3M). Resulting films were then incubated for at least48 hours at 32° C. before enumeration using the petrifilm reader.

Sonicated Counts Results

FIGS. 42 and 43 show the results of the sonicated counts. A largerreduction in surface count was seen against S. epidermidis than eMRSA 15(epidemic MRSA 15, a UK hospital isolate) though grow back was seen atthe 72 hour timepoint to levels equivalent to the initial inoculationand the equivalent control.

Counts From Suspension

After the appropriate time period ImI of the inoculum was sampled fromreps 1 to 3 (of all samples and added to 9 ml of STS). ImI of this wasthen plated in duplicate and ImI was serially diluted, to 10″ at time 0,10″ at 24 hours and 10″ thereafter for negative controls, and 10″ at alltime points for coated samples and positive controls, in MRD.

For uncoated samples and culture controls at 0 and 24 hours dilutionsfrom 10″² downwards and 10″³ downwards respectively were plated out onduplicate Aerobic Count Petrifilm (3M). For coated samples all dilutionswere plated in duplicate on Aerobic Count Petrifilm (3M). The resultingplates were incubated at 32° C. for at least 48 hours before counting.

Suspension Counts Results

FIGS. 44 and 45 show the results of the suspension counts. Counts fromthe suspension can be used to calculate a reduction in count from time 0and therefore a log reduction. Against eMRSA 15 the lower dose silvershowed little kill but did keep numbers in suspension static. The highersilver dose killed to the limit of detection and maintained numbersthere. Both doses of silver HA initially killed S. epidermidis down to alow level but there was grow back of the organism to levels comparablewith the initial organism at 72 hours.

Example 10

Cytotoxicity testing of VPS Ag/HA samples

The coatings from Example 2 were evaluated for cytotoxicity. Inaddition, uncoated Ti6A14V coupons (negative control known to benon-cytotoxic) were also evaluated. r002821 Method

Materials Uncoated Ti6A14V coupons VPS-Hydroxyapatite (HA) coupons—BatchTO035A VPS-HA with low dose silver (1%) (HAD—Batch TO035B VPS-HA withmedium dose silver (2%) (HA2)—Batch TO035C Polyvinyl chloridediscs—Batch TO024-90-02-01 High Density Polyethylene discs—BatchTO024-90-02-02

Alpha—Minimum Essential Medium Eagles (alpha-MEM) plus 10% foetal calfserum-#9696, #9662, #9707

Trypsin-EDTA—Lot 058K2373

Trypan Blue—Sigma T8154 Lot 088k2379 and Lot 047k2349

Vectashield mounting medium for fluorescence with DAPI-Vector H-1200 LotU0403 WST-I reagent-Roche 11644807001 Lot 14473200

Preparation of Conditioned Media

Using ISO10993 guidelines the surface area to volume ratio wascalculated for all the test and control samples to determine the volumeof media required per sample to produce liquid extracts of the samples(referred to as conditioned media). For the HA, HA1, HA2 and titaniumcoupons the volume of media required was 1.23 ml/coupon, four couponswere placed in a well of a 6-well plate (n=4) in a total volume of 4.92ml. The volume of media required for the PVC and HDPE discs was 1.33ml/disc, four discs were placed in a well of a 6-well plate (n=4) in atotal volume of 5.32 ml. The layout of these groups in the 6-well plateswas randomised to help reduce the effect of plate layout. Alongsidethese plates a 6-well plate had 5.32 ml/well of media added, acting asthe tissue culture plastic group (TCP) as this group is only used as anassay control and therefore did not need to be included in therandomisation. The media incubated with the test materials wasalpha-MEM; these five plates were incubated at 37° C., 5% CO2 for 7days.

Cell Seeding

MC3T3-E1 cells were passaged for counting according to SOP/CB/006. Thecells were required for seeding at 5×10/cm . 168 wells were seeded over2×96-well plates at this density, the cells were now P13. Cells werecultured for 48 hours at 37° C., 5% CO2.

Application of Conditioned Media

The media was removed from the 96-well plates and replaced with 100 μlof the appropriate conditioned media, n=6 per sample group pertreatment/control group. The 2×96-well plates were incubated for24-hours at 37° C., 5% CO2. r002901 WST-I Assay

WST-I reagent is used to quantify the metabolic activity of the cellsexposed to the conditioned media. 10 μl/well of WST-I reagent was addedto each of the 96-well plates. The plates were incubated for 1 hour at37° C., 5% CO2 shaken for 1 minute and then read on the Multiskan platereader (asset no. 00005247) at 450 nm and 650 nm for the test andreference wavelengths respectively. The reference wavelength wassubtracted from the test wavelength for each well and the means for eachgroup calculated and plotted to demonstrate the metabolic activity ofthe cells (see FIG. 46).

Results and Discussion

Mycoplasma Testing

Mycoplasma testing by DAPI staining was conducted according toSOP/CB/069. The cells used in this experiment were deemed mycoplasmanegative.

WST-I Assay

The HA1 and HA2 groups stimulated increased metabolic activity ofMC3T3-E1 cells in this experiment compared to the positive cytotoxiccontrol group PVC, and the negative control group HDPE. The metabolicactivity of cells exposed to HA alone was however higher than either ofthe two Ag containing groups. The metabolic activity of cells exposed tothe Ti6A14V group was below that of all HA containing groups and thenegative control HDPE. H6A14V did however stimulate the mineralisationof cells to a level that was twice that of the positive control. The TCPgroup was used as cell control group and cells in this group metabolisedat a similar level to the negative control HDPE. It was noted that themetabolic activity of cells in the titanium group was below that of thetwo negative controls however there was a large spread of data from thisdata set as shown by the error bars.

Conclusions

VPS Ag/HA samples at 1 and 2% silver increased the metabolic activity ofMC3T3-E1 cells to levels above that of the positive and negativecontrols. However, the metabolic activity of cells from these groups waslower than cells in the HA alone group.

The skilled person will realize that the breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments but should be defined only in accordance with anyclaims appended hereto and their equivalents.

1-51. (canceled)
 52. A medical implant comprising an implant surfacehaving a surface coating comprising an osseointegration agent, and anantimicrobial metal agent present in discrete particles and at aconcentration sufficient to have an anti-bacterial effect.
 53. Themedical implant of claim 52, wherein the antimicrobial metal agent inthe surface coating is present at about 0.1 to about 10 weight percent.54. The medical implant of claim 52, wherein in the antimicrobial metalagent is silver, copper, or zinc.
 55. The medical implant of claim 52,wherein the discrete particles of an antimicrobial metal agent areparticles of metallic silver or a silver compound.
 56. The medicalimplant of claim 52, wherein the discrete particles are discretemetallic silver particles.
 57. The medical implant of claim 56, whereinthe metallic silver particles are spherical or irregular in shape. 58.The medical implant of claim 57, wherein the diameter of the metallicsilver particles is about 15 nm to about 10 μm.
 59. The medical implantof claim 52, wherein an osseointegration agent is substituted with anantimicrobial metal agent.
 60. The medical implant of claim 54, whereinan osseointegration agent is substituted with one or more of theantimicrobial metal agents silver, copper, or zinc.
 61. The medicalimplant of claim 56, wherein an osseointegration agent issilver-substituted.
 62. The medical implant of claim 61, wherein thesilver concentration from the silver-substituted osseointegration agentis substantially homogeneous throughout the surface coating.
 63. Themedical implant of claim 62, wherein the silver-substitutedosseointegration agent contains about 0.1 to about 10% by weight ofsilver.
 64. The medical implant of claim 62, wherein thesilver-substituted osseointegration agent contains about 0.5 to about3.0% by weight of silver.
 65. The medical implant of claim 61, whereinthe osseointegration agent is a calcium derivative.
 66. The medicalimplant of claim 65, wherein the osseointegration agent ishydroxyapatite, β-tricalcium phosphate, or a mixture of both.
 67. Themedical implant of claim 52, wherein the thickness of the surfacecoating is about 1 μm.
 68. The medical implant of claim 52, wherein thethickness of the surface coating is about 10 μm to about 200 μm.
 69. Themedical implant of claim 52, wherein the thickness of the surfacecoating is about 30 μm to about 100 μm.
 70. The medical implant of claim52, wherein the tensile attachment strength of the surface coating tothe implant surface is equal to or greater than about 15 MPa.
 71. Themedical implant of claim 52, wherein the surface coating further hasosteoinductive, or osteopromotive properties.
 72. The medical implant ofclaim 52, wherein the osteointegration agent is substituted with atleast one of the following, silver, carbonate, fluoride, silicon,magnesium, strontium, vanadium, lithium, copper, or zinc.
 73. A processfor making the medical implant of claim 66, comprising the steps ofpreparing a silver-substituted calcium derivative osseointegration agentby an ion exchange or sol-gel process, obtaining silver substitutedcalcium derivative osseointegration agents in powder form that areapproximately the same size and shape as nonsilver-substituted calciumderivative osseointegration agents in powder form, applying a surfacecoating to a medical implant comprising an implant surface by plasmaspraying the implant surface while in a reducing environment, obtaininga medical implant having a surface coating comprising silver-substitutedosseointegration agents hydroxyapatite, β-tricalcium phosphate, ormixtures of both, and antimicrobial metal agents present as discretemetallic silver particles at a concentration sufficient to have ananti-bacterial effect.